Cardiac progenitor cells (CPCs) in the adult mammalian heart are identified by stem cell-related antigens, multipotency, and by their ability to regenerate myocardium endogenously and iatrogenically. CPCs, when injected into the heart after ischemic injury, produce limited multilineage differentiation (cardiomyocytes, endothelial cells, vascular smooth muscle cells) and functional benefits. The origin of CPCs is as-yet unclear. Some if not most CPCs are likely of innate embryonic origin within the heart; exogenous bloodborne precursor cells can also seed the heart after injury. Another potential mechanism of origin that has been overlooked is that of dedifferentiation, a process whereby mature, specialized cells regain proliferative ability and augmented plasticity. The established paradigm for cardiomyocyte development posits a one-way transit from precursor cells through neonatal cardiomyocytes on to mature heart cells, which can no longer divide and proliferate. Adult zebrafish cardiomyocytes, in contrast, re-enter the cell cycle with alacrity, but even they have not been demonstrated to dedifferentiate to a state of multipotency. The major discovery in the initial grant period of this R01 was the recognition of the ability of postnatal mammalian cardiomyocytes to dedifferentiate and acquire features of CPCs. Preliminary data supporting this claim are presented in the progress report. The proposed ability of dedifferentiated cardiomyocytes to become CPCs, if verified by the work proposed here, would change fundamentally our view of the adult heart and its potential plasticity. For this reason, we seek to demonstrate cardiomyocyte dedifferentiation to CPCs by multiple complementary experimental approaches; to characterize the underlying molecular and cellular mechanisms; and to establish the pathophysiological relevance of dedifferentiation. We will test the idea that cell culture promotes dedifferentiation, cell cycle re- entry, and acquisition of stemness. Genetic cell fate mapping will be utilized to establish that confirmed cardiomyocytes dedifferentiate, express c-kit, and are capable of differentiating into at least two cardiac lineages. Specific questions to be addressed include: How often and under what conditions do dedifferentiating cardiomyocytes re-enter the cell cycle? What changes in gene expression, microRNAs, and cell cycle regulatory proteins underlie the dedifferentiation process? Are autocrine/paracrine factors in conditioned media important in cardiomyocyte dedifferentiation? Do dedifferentiated cardiomyocytes express stem cell antigens? Are they pluripotent? Can they be clonally proliferated? Can dedifferentiated cardiomyocytes re-differentiate and transdifferentiate in vitro? When injected into injured hearts, do dedifferentiated cardiomyocytes engraft, differentiate and improve function? Do genetically-defined cardiomyocytes dedifferentiate in situ in response to injury? Does endogenous myocardial regeneration after infarction reflect dedifferentiation and subsequent multilineage differentiation of cardiomyocytes? The work tests the idea that CPCs may arise from dedifferentiation as well as embryogenesis or bloodborne seeding. PUBLIC HEALTH RELEVANCE: The proposed work focuses on the heart's innate ability to regenerate, and how that ability might be tapped to improve therapies for heart disease. We seek to understand the mechanism of origin of innate progenitor cells in the heart that normally function to repair the wear and tear of daily life. We will test the idea that mature heart cells can go backwards and regain the features of innate progenitor cells, an idea which has important biological, pathophysiological and therapeutic implications.